Cardiovascular disease, including coronary heart disease caused by atherosclerosis, is the single largest killer of adults in North America (2002 Heart and Stroke Statistical Update). The development and progression of atherosclerosis in coronary arteries can lead to heart attacks and angina. In 1999 it was estimated that 12.6 million Americans had coronary heart disease. Approximately 1 in 5 deaths in 1999 were due to coronary heart disease, with a total US and Canadian mortality of over 500,000 and 42,000 individuals, respectively. It is estimated that over 102 million American adults have blood cholesterol levels that are either border-line high risk, or high risk of developing coronary heart disease. In addition to the immediate social and economic burden that heart attacks have on our health care system, there also is the considerable cost associated with the aftermath of a coronary heart disease event. About 25% of males and 38% of females will die one year after a heart attack, and death by coronary heart disease tends to occur during a person's peak productive years (BRFSS [1997], MMWR vol. 49, No. SS-2, Mar. 24, 2000, CDC/NCHS). There is also a further economic burden of coronary heart disease associated with premature and permanent disability of the labor force. In 1998, over $10 billion was paid to Medicare beneficiaries for coronary heart disease (Health Care Financing Review, Statistical Supplement [2000], HFCA).
Patients currently have a choice of a number of different drugs to treat cardiovascular disease/coronary heart disease. These drugs fall into various classes, including antihypertensives and antihyperlipidemics. Although these products have been shown to be beneficial in reducing the progression of coronary heart disease and preventing heart attacks, they can be limited in their effectiveness in some individuals because of low tolerability and, in some cases, mitigation of drug efficacy by the compensatory effects of the liver (Turley, S. D. (2002) Am. J. Managed Care 8 (2 Suppl):S29-32).
The accumulation of lipids, especially cholesterol, in several aortic and arterial cell-types, such as macrophages and smooth muscle cells, is the defining pathologic feature of atherosclerosis (Gotlieb et al. (1999) Blood Vessels. In Pathology. Rubin, E. and Farber, J. L., editors. Lippincott-Raven, Philadelphia, New York. 481-530). Major investigative efforts are being expended to understand two central issues related to this problem. The first relates to the mechanism by which cholesterol is delivered to, and taken up by, these cells. The second relates to the process by which these cells export and rid themselves of excessive cholesterol. In the treatment and prevention of atherosclerosis, one of the aims is to limit the intracellular accumulation of large quantities of cholesterol that adversely influence the viability of these cells, thereby eventually altering the structural integrity of the blood vessels.
An analogous set of events occurs in acute tissue injury. Such injuries result in local cell death and set in motion local inflammation and the systemic acute phase response (Fantone, J. C. and Ward, P. A. (1994) Inflammation. In Pathology. Rubin, E. and Farber, J. editors. Lippincott, Philadelphia. 32-6). Alterations in local cholesterol processing are important components of this process. At sites of acute tissue injury, dying cells release large quantities of cell debris that includes cell membrane fragments rich in cholesterol (Fantone, J. C. and Ward, P. A. (1994) Inflammation. In Pathology. Rubin, E. and Farber, J. editors. Lippincott, Philadephia. 32-6). As part of acute inflammation, macrophages arriving at sites of injury ingest these fragments for further processing and thereby acquire a considerable cholesterol load, becoming foam cells, analogous to those seen in atherosclerosis. During acute tissue injury and the consequent acute inflammatory process, a cholesterol removal mechanism is required to mobilize the cholesterol either for excretion or re-use.
The physiological role of one of the major acute phase (AP) proteins synthesized by the liver in response to tissue injury, serum amyloid A (SAA), is directly related to these events and processes. SAA represents a group of four polymorphic proteins, encoded by a multigene family, that have been conserved for over 600 million years (Jensen et al. (1997) J. Immunol. 158:384-392; Santiago et al. (2000) J. Exp. Zool. 288: 3335-344). Isoforms SAA1.1 and SAA2.1 are present in plasma in acute phase tissue injury and are the most thoroughly investigated.
The nomenclature for serum amyloid A was revised in 1999, as there was a recognized need by researchers for a systematic nomenclature of the multiple SAA genes in human and animal models and for their allelic variants (Amyloid: Int. J. Exp. Clin. Invest. 1999 6:67-70). The major revision was the re-designation of the mouse Saa1 and Saa2 genes. Based upon chromosomal mapping, it appears that the mouse Saa2 locus corresponds to human SAA1. Therefore, the mouse nomenclature was changed to be fully compatible with the human nomenclature.
The following Tables set forth the revised nomenclature for SAA mouse and human proteins as well as their corresponding sequences. These tables are based upon the disclosure in 1999 in Amyloid: Int. J. Exp. Clin. Invest. 6:67-70. The tables presented herein have been modified, however, to clarify alignment and provide numbering for residue (−1) of mouse isoform SAA3 comprising an additional amino acid.
TABLE IMouse SAA ProteinsSeqNewOldID #−11234567891011121314SAA1.1SAA218GFFSFIGEAFQGAGSAA1.2SJL/J33SAA1.3mc134SSAA1.4mc235SSAA1.5mm136VHSAA1.6mm237SAA2.1SAA119VHSAA2.2CE/J38VHLSAA339QRWVQMKGSRSAA4SAA540DWYFRTW15161718192021222324252627282930SAA1.1SAA2DMWRAYTDMKEAGWKDSAA1.2SJL/JSAA1.3mc1RSAA1.4mc2SAA1.5mm1SAA1.6mm2SAA2.1SAA1NNSAA2.2CE/JSAA3SKSAA4SAA5LRNLNYQN31323334353637383940414243444546SAA1.1SAA2GDKYFHARGNYDAAQRSAA1.2SJL/JSAA1.3mc1RSAA1.4mc2SAA1.5mm1SAA1.6mm2SAA2.1SAA1SSAA2.2CE/JSAA3SRSAA4SAA5AQYEQ5 47484950515253545556575859606162SAA1.1SAA2GPGGVWAAEKISDARESAA1.2SJL/JSAA1.3mc1SAA1.4mc2SAA1.5mm1SAA1.6mm2SAA2.1SAA1GSAA2.2CE/JSAA3AKVSAA4SAA5SIKITSK63646566676869707172737475767778SAA1.1SAA2SFQEFFGRGHEDTMADSAA1.2SJL/JSAA1.3mc1SAA1.4mc2GSAA1.5mm1SAA1.6mm2SAA2.1SAA1AISAA2.2CE/JASAA3VKTHASRSAA4SAA5YGLLNHLTLQT  ↑[NRYYFGIR]79808182838485868788899091929394SAA1.1SAA2QEANRHGRSGKDPNYYSAA1.2SJL/JSAA1.3mc1SAA1.4mc2SAA1.5mm1SAA1.6mm2SAA2.1SAA1SAA2.2CE/JSAA3FEWHFSAA4SAA5KEEWNHF9596979899100101102103SAA1.1SAA2RPPGLPAKYSAA1.2SJL/JDSAA1.3mc1SAA1.4mc2SAA1.5mm1SAA1.6mm2DSAA2.1SAA1DSAA2.2CE/JDSAA3AKRSAA4SAA5EEF
TABLE IIHuman SAA proteinsSeqNewOldID#123456789101112131415SAA1.1SAA1α20RSFFSFLGEAFDGARSAA1.2SAA1β41SAA1.3SAA1γ42SAA1.4SAA1δ43SAA1.5SAA1β44SAA2.1SAA2α21SAA2.2SAA2β45SAA446ESWRSFFKEALQGVG16171819202122232425262728293031SAA1.1SAA1αDMWRAYSDMREANYIGSAA1.2SAA1βSAA1.3SAA1γSAA1.4SAA1δSAA1.5SAA1βSAA2.1SAA2αSAA2.2SAA2βSAA4DMGRAYMDIMISMHQN32333435363738394041424344454647SAA1.1SAA1αSDKYFHARGNYDAAKRSAA1.2SAA1βSAA1.3SAA1γSAA1.4SAA1δSAA1.5SAA1βSAA2.1SAA2αSAA2.2SAA2βSAA4SNRYLYARGNYDAAQR48495051525354555657585960616263SAA1.1SAA1αGPGGVWAAEAISDARESAA1.2SAA1βAVSAA1.3SAA1γASAA1.4SAA1δAVNSAA1.5SAA1βAVSAA2.1SAA2αAVNSAA2.2SAA2βAVNSAA4GPGGVWAAKLISRSRV64656667686970717273747576777879SAA1.1SAA1αNIQRFFGHGAEDSLADSAA1.2SAA1βDSAA1.3SAA1γSAA1.4SAA1δSAA1.5SAA1βSAA2.1SAA2αLTSAA2.2SAA2βLTRSAA4YLQGLISTVLEDSKSN  ↑[DYYLFGNS]80818283848586878889909192939495SAA1.1SAA1αQAANEWGRSGKDPNHFSAA1.2SAA1βSAA1.3SAA1γSAA1.4SAA1δSAA1.5SAA1βSAA2.1SAA2αKRSAA2.2SAA2βKRSAA4EKAEEWGRSGKDPDRF96979899100101102103104SAA1.1SAA1αRPAGLPEKYSAA1.2SAA1βSAA1.3SAA1γSAA1.4SAA1δSAA1.5SAA1βSAA2.1SAA2αSAA2.2SAA2βSAA4RPDGLPKKYThe nomenclature for SAA proteins employed in this patent application corresponds to the revised nomenclature as set forth in the above Tables. However, it must be appreciated that journal references published prior to this 1999 revision and patent applications filed prior to this 1999 revision may use the old nomenclature, thus, for example referring to mouse Saa1 as mouse Saa2 and vice versa.
SAA isoforms SAA1.1 and SAA2.1 are produced primarily by hepatocytes in response to various causes of tissue injury and inflammation (Morrow et al. (1981) Proc. Natl. Acad. Sci. USA 78:4718-4722). Synthesis of SAA1.1 and 2.1 by the liver is induced by cytokines such as interleukin-1, interleukin-6, and tumor necrosis factor, which are released by activated macrophages, and which act through a set of downstream effectors in the hepatocyte cytoplasm and nucleus (Edbrooke et al. (1991) Cytokine 3:380-388; Betts et al. (1993) J. Biol. Chem. 268:25624-25631; Ray et al. (1999) J. Biol. Chem. 274:4300-430810; and Sipe et al. (1987) Lymphokine Res. 6:93-101). Maximum transcription rates for the SAA1.1 and 2.1 genes are seen 3-4 hours following tissue injury, and within 18-24 hours of injury the plasma concentration of these two proteins rises from 1-5 μg/mL to 500-1000 μg/mL (500-1000-fold increase) (McAdam et al. (1978) J. Clin. Invest. 61:390-394; McAdam, K. P., Sipe, J. D. (1976) J. Exp. Med. 144:1121-1127). Once secreted from hepatocytes, SAA1.1 and 2.1 are found predominantly in the high density lipoprotein (HDL) fraction and form 30-80% of the HDL apolipoproteins, resulting in a major reorganization of the apolipoprotein composition of the HDL fraction (Benditt et al. (1979) Proc. Natl. Acad. Sci. USA 76: 4092-4096; Hoffman, J. S. and Benditt, E. P. (1982) J. Biol. Chem. 257:10518-10522).
At present there is debate whether the observed increase in SAA expression during tissue injury is associated with a beneficial role against atherosclerotic lesions, or whether increased SAA levels are in fact associated with a role in developing atherosclerosis.
Elevated levels of SAA isoforms are observed during the early pathological vascular events leading to atherosclerosis before clinical symptoms are evident. (reviewed in Kisilevsky, R. and Tam, S.-P. (2002) Pediatric Pathol. and Mol. Med. 21: 291-303). This elevation has led some researchers to suggest that SAA levels may play a causative, contributing role in atherogenesis (Jousilahti et al. (2001) Atherosclerosis 156:451-456; Kumon et al. (1998) Scand. J. Immunol. 48:419-424; Liuzzo et al. (1994) N. Engl. J Med. 331:417-424; Ridker et al. (1998) Circulation 98:839-844; Rosenthal, C. J. and Franklin, E. C. (1975) J. Clin, Invest. 55:746-753; Steinmetz et al. (1989) Biochim Biophys. Acta. 1006:173-178; Van Lenten et al. (1995) J. Clin. Invest. 96:2758-2767).
However, there have also been reports of SAA and isoforms thereof promoting the efflux of cholesterol from macrophages.
For example, high density lipoprotein-serum amyloid A (HDL-SAA) has been shown to have reduced ability to accept cholesterol from low density lipoprotein/very low density lipoprotein (LDL/VLDL), ensuring that HDL, in its afferent route, arrives at macrophages carrying as little cholesterol as possible (Kisilevsky et al. (1996) Amyloid 3: 252-260). Thus, this form of HDL has a greater capacity to accept cholesterol from cholesterol-laden macrophages. HDL-SAA has also been demonstrated to have a 3 to 4-fold higher affinity for macrophages when compared to HDL alone. Further, an increase was observed in the number of HDL-SAA binding sites on macrophages obtained from animals with an AP inflammatory reaction. Competition studies with macrophages (Kisilevsky, R. and Subrahmanyan, L. (1992) Lab. Invest. 66: 778-785) showed that unlabelled HDL-SAA, but not HDL alone, effectively displaced radiolabeled HDL-SAA. This preferential displacement by HDL-SAA is likely indicative of the presence of SAA receptors on the macrophages. Such SAA receptors are separate and additional to the binding sites for apoA-1 on the macrophages (Kisilevsky, R. and Subrahmanyan, L. (1992) Lab. Invest. 66: 778-785; U.S. Pat. No. 6,004,936). The presence of SAA receptors is further supported by the demonstration that HDL-SAA was in clathrin coated pits shortly after binding to macrophages. These pits and the resulting endosomes are consistent with the concept of receptor-mediated endocytosis, a process that is dependent on cell surface heparin sulphate, to which SAA binds effectively (Ancsin, J. and Kisilevsky, R. (1999) J. Biol. Chem. 274:7172-7181; Rocken, C. and Kisilevsky, R. (1997) Amyloid 4: 259-273).
More recent studies have demonstrated that SAA enhances HDL uptake by macrophages (Banka et al. (1995) J. Lipid Res. 36:1058-10865) and has an affinity for cholesterol (Liang, J. S. and Sipe, J. D. (1995) J. Lipid Res. 36:37-46). Using synthetic peptides corresponding to residues 1-18 and 40-63 of human apoSAA1 (now referred to as SAA1.1) and residues 1-18 of human apoSAA4 (now referred to as SAA4) it was shown that apoSAA1 but not apoSAA4 binds cholesterol at the amino terminal region (Liang et al. (1996) J. Lipid Res. 37:2109-2116).
Furthermore murine SAA2.1, but not murine SAA1.1, was shown to inhibit macrophage acyl CoA:cholesterol acyl transferase (ACAT) activity in culture in intact murine macrophages and in their post-nuclear homogenates in a dose-dependent manner (Ely et al. (2001) Amyloid 8:169-181). Further examination of cyanogen bromide generated cleavage fragments of murine SAA2.1 purified by reverse phase HPLC showed murine SAA2.11-16 to have a profound effect inhibiting ACAT activity in a dose-dependent manner. In contrast, murine SAA2.124-103 exhibited no inhibitory effect on ACAT activity (Ely et al. (2001) Amyloid 8:169-181).
Murine SAA2.1 has also been shown to stimulate hepatic, macrophage, and pancreatic cholesterol esterase activities in vitro (Lindhorst et al. (1997) Biochim. Biophys. Acta 1339:143-154; Ely et al. (2001) Amyloid 8:169-181; Tam et al. (2002) J. Lipid Res. 43:1410-1420). This effect was shown to reside in the 80 residue COOH-terminal region of murine SAA2.1 liberated by cyanogen bromide cleavage (Ely et al. (2001) Amyloid 8:169-181). This 80 residue region comprises residues 24-103 of murine SAA2.1.
The ability of HDL-SAA and liposomes containing murine SAA2.1 to cause a marked reduction of acyl CoA:cholesterol acyl transferase activity and enhancement of cholesterol efflux activity was confirmed in macrophages in culture (Tam et al. (2002) J. Lipid Res. 43:1410-1420). Intravenous injection of [3H]-cholesterol-loaded macrophages into inflamed mice has also been reported to result in a 3- to 3.5-fold increase in the amount of radiolabeled cholesterol released into the plasma when compared to similarly treated un-inflamed control animals (Tam et al. J. Lipid Res. 2002 43:1410-1420). In this study, macrophage cholesterol efflux was shown to be coupled to the ATP-binding cassette transporter, ABCA1, which is an important protein for the initial step of the reverse cholesterol transport pathway. Furthermore, [3H]-cholesterol-laden macrophages, when pre-treated with HDL-SAA2.1 (murine) in tissue culture and then injected into un-inflamed mice, rapidly released their cholesterol into the plasma (Tam et al. (2002) J. Lipid Res. 43:1410-1420). This result was not observed when macrophages were treated with HDL alone.
Thus, isoforms SAA1.1 and SAA2.1 are up-regulated during inflammation; they are evolutionarily conserved; and they are predominantly associated with HDL and HDL's established role in the reverse cholesterol transport pathway (Lindhorst et al. (1997) Biochim. Biophys. Acta 1339:143-154; Kisilevsky, R. (1991) Med. Hypotheses 35: 337-341; Kisilevsky, R. et al. (1996) Amyloid 3: 252-260; and Kisilevsky, R. and Subrahmanyan, L. (1992) Lab. Invest. 66: 778-785).
U.S. Pat. No. 5,318,958 discloses methods of potentiating the release and collection of macrophage cholesterol in vivo by administering an effective amount of HDL bound to a ligand having serum amyloid A affinity for HDL. A preferred ligand of this method taught in this patent is serum amyloid A itself.
U.S. Pat. No. 6,004,936 describes similar methods to U.S. Pat. No. 5,318,958. However, in the method claimed in U.S. Pat. No. 6,004,936, the ligand having serum amyloid affinity is not bound to HDL prior to administration. This patent teaches that preferred ligands having serum amyloid affinity are non-amyloidogenic isoforms of serum amyloid A such as SAA2.1.